Regulation of Alternative Splicing of Lipid Metabolism Genes in Sepsis-Induced Liver Damage by RNA-Binding Proteins.

RNA binding proteins (RBPs) have the potential for transcriptional regulation in sepsis-induced liver injury, but precise functions remain unclear. Our aim is to conduct a genome-wide expression analysis of RBPs and illuminate changes in the regulation of alternative splicing in sepsis-induced liver injury. RNA-seq data on "sepsis and liver" from the publicly available NCBI data set was analyzed, and differentially expressed RBPs and alternative splicing events (ASEs) in the healthy and septic liver were identified. Co-expression analyses of sepsis-regulated RBPs and ASEs were performed. Models of sepsis were established to validate hepatic RBP gene expression patterns with different treatments. Pairwise analysis of gene expression profiles of sham, cecum ligation puncture (CLP), and CLP with dichloroacetate (CLPDCA) mice allowed 1208 differentially expressed genes (DEGs), of which 800 were up-regulated and 408 down-regulated, to be identified. DEGs were similar in both Sham and CLPDCA mice. The KEGG analysis showed that up-regulated genes as being involved in cytokine-cytokine receptor interaction and IL-17 signaling pathway and down-regulated genes in metabolic pathways. Differences in lipid metabolism-related alternative splicing events, including A3SS, were also found in CLP and CLPDCA compared with sham mice. Thirty-seven RBPs, including S100a11, Ads2, Fndc3b, Fn1, Ddx28, Car2, Cisd1, and Ptms, were differentially expressed in CLP mice and the regulated alternative splicing genes(RASG) with the RBP shown to be enriched in lipid metabolic and oxidation-reduction-related processes by GO functional analysis. In KEEG analysis the RASG mainly enriched in metabolic pathway. The models of sepsis were constructed with different treatment groups, and S100a11 expression in the CLP group found to be higher than in the sham group, a change that was reversed by DCA. The alternative splicing ratio of Srebf1 and Cers2 decreased compared with the sham group increased after DCA treatment. Abnormal profiles of gene expression and alternative splicing were associated with sepsis-induced liver injury. Unusual expression of RBPs, such as S100a11, may regulate alternative splicing of lipid metabolism-associated genes, such as Srebf1 and Cers2, in the septic liver. RBPs may constitute potential treatment targets for sepsis-induced liver injury.

Identification of heat shock protein family A member 5 (HSPA5) targets involved in nonalcoholic fatty liver disease.

Heat shock protein family A (Hsp70) member 5 (HSPA5) is an endoplasmic reticulum chaperone, which regulates cell metabolism, particularly lipid metabolism. While HSPA5's role in regulating cell function is well described, HSPA5 binding to RNA and its biological function in nonalcoholic fatty liver disease (NAFLD) is still lacking. In the present study, the ability of HSPA5 to modulate alternative splicing (AS) of cellular genes was assessed using Real-Time PCR on 89 NAFLD-associated genes. RNA immunoprecipitation coupled to RNA sequencing (RIP-Seq) assays were also performed to identify cellular mRNAs bound by HSPA5. We obtained the HSPA5-bound RNA profile in HeLa cells and peak calling analysis revealed that HSPA5 binds to coding genes and lncRNAs. Moreover, RIP-Seq assays demonstrated that HSPA5 immunoprecipitates specific cellular mRNAs such as EGFR, NEAT1, LRP1 and TGFß1, which are important in the pathology of NAFLD. Finally, HSPA5 binding sites may be associated with splicing sites. We used the HOMER algorithm to search for motifs enriched in coding sequence (CDs) peaks, which identified over-representation of the AGAG motif in both sets of immunoprecipitated peaks. HSPA5 regulated genes at the 5'UTR alternative splicing and introns and in an AG-rich sequence-dependent manner. We propose that the HSPA5-AGAG interaction might play an important role in regulating alternative splicing of NAFLD-related genes. This report is the first to demonstrate that HSPA5 regulated pre-RNA alternative splicing, stability, or translation and affected target protein(s) via binding to lncRNA and mRNA linked to NAFLD.

RALY regulate the proliferation and expression of immune/inflammatory response genes via alternative splicing of FOS.

RALY is a multifunctional RNA-binding protein involved in cancer metastasis, prognosis, and chemotherapy resistance in various cancers. However, the molecular mechanism of which is still unclear. We have established RALY overexpression cell lines and studied the effect of RALY on proliferation and apoptosis in HeLa cells. Then we used RNA-seq to analyze the transcriptomes data. Lastly, RT-qPCR experiments had performed to confirm the RNA-seq results. We found that the overexpression of RALY in HeLa cells inhibited proliferation. Moreover, the overexpression of RALY changed the gene expression profile, and the significant upregulation of genes involved immune/inflammatory response related biological process by NOD-like receptor signaling pathway cytokine-cytokine receptor interaction. The significant downregulation genes involved innate immune response by the Primary immunodeficiency pathway. Notably, IFIT1, IFIT2, IFTI3, IFI44, HERC4, and OASL expression had inhibited by the overexpression of RALY. Furthermore, RALY negatively regulates the expression of transcription factors FOS and FOSB. Notably, we found that 645 alternative splicing events had regulated by overexpression of RALY, which is highly enriched in transcription regulation, RNA splicing, and cell proliferation biological process by the metabolic pathway. We show that RALY regulates the expression of immune/inflammatory response-related genes via alternative splicing of FOS in HeLa cells. The novel role of RALY in regulating immune/inflammatory gene expression may explain its function in regulating chemotherapy resistance and provides novel insights into further exploring the molecular mechanism of RALY in regulating cancer immunity and chemo/immune therapies.

Preparation and identification of an anti-idiotypic antibody antagonist (FG8) for EGFR that shows potential activity against liver cancer cells.

OBJECTIVE: Currently, there are two categories of epidermal growth factor receptor (EGFR) antagonists, small molecule antagonists and anti-EGFR antibodies. In the current study, we developed a new EGFR antagonist employing the anti-idiotypic antibodies strategy. RESULTS: First, using EGF as an antigen, through a series of immunological protocols and hybridoma technology, we obtained an anti-idiotypic antibody against EGF receptor-binding epitopes. On this basis, we screened and characterized the anti-idiotype antibodies against EGFR through competitive ELISA, co-localization analysis, competitive receptor binding analysis, and immunofluorescence. Finally, an internal image anti-idiotype antibody called FG8 was successfully prepared. Experiment result shows that FG8 inhibits EGFR-mediated signaling pathways in vitro. Additionally, FG8 inhibits liver tumor cell proliferation as well as induces tumor cell apoptosis. CONCLUSIONS: The present study suggests that FG8 is a potential therapeutic agent for liver cancer. In addition, this study provides a novel method for the preparation of EGFR antagonists.

A novel function of CYP21A2 in regulating cell migration and invasion via Wnt signaling.

CYP21A2, which is responsible for 21-hydroxylase activity, is prominent to the development of congenital adrenal hyperplasia (CAH). The aim of our current study is to investigate the role of CYP21A2 in the tumor processes. Here, we used HepG2 cell lines and generated CYP21A2 overexpressing vector and siRNA to investigate the effect of CYP21A2 on the tumor development processes, particularly cell migration and invasion; genes expression related to these processes were further examined. Results showed that CYP21A2 over-expressed or silenced had no effects on cell viability as well as the process of cell apoptosis. Further study suggested that CYP21A2 silenced significantly decreased the G0/G1 phase and increased the S phase of the cell cycle. However, no differences were observed when CYP21A2 was overexpressed. Moreover, we found that cell migration and invasion significantly improved with CYP21A2 overexpressed and impaired with silenced CYP21A2. Finally, we examined the expression of genes related to tumor processes and found that the Wnt signaling genes were changed. Taken together, our results demonstrated a novel function of CYP21A2 in the regulation of tumor processes, particularly cell migration and invasion, which this may be mediated by the Wnt signaling pathway.